Using the cDNA clones for Alpha-lactalbumin, whey phosphoprotein and Kappa-protein, we have screened a bacteriophage Charon 4A/rat partial EcoRI genomic library and isolated phages containing genomic DNA corresponding to these cDNA's. The genomic maps for the three individual genes have been established from the phages carrying overlapping DNA fragments. Entire Alpha-lactalbumin gene sequence has been determined and compared with the chicken lysozyme gene, since it was proposed that the two genes have arisen from a common ancestral gene. These results show that: a) the 5'-flanking sequence of Alpha-lactalbumin gene contains almost identical short repeat sequences; b) a nanonucleotide sequence ATCCCTTTC is repeated 3 times which resembles with the part of 19 nucleotide concensus sequence ATC(CC) ATTT(A)TCTG(G)TTGTA thought to be involved in the progesterone receptor recognition site in ovalbumin gene; c) both, Alpha-lactalbumin and lysozyme genes contain 3 introns at similar positions; d) the first three exons of the two genes show high nucleotide homologies and are of comparable lengths and e) the fourht exon of Alpha-lactalbumin, which codes for the amino acid residues essential for its interaction with galactosyltransferase, is markedly different from the corresponding exon of lysozyme and is precede by two (TG)n repeats, (TG)25 and (TG)22. It is suggested that the 4th exon of Alpha-LA coding for a new functional unit, might have replaced the DNA region of a primordial lysozyme gene and led to a protein with a new function.